PROJECT SUMMARY/ABSTRACT Neuromodulators, such as dopamine and opioids, dynamically regulate synaptic strength and intrinsic properties of neurons, thereby modulating outputs of neural circuits and modifying behavior. Their alteration is linked to devastating diseases such as Parkinson's Disease and addiction. The striatum is an ideal system to study neuromodulation since it receives a large number of behaviorally-relevant neuromodulators. In addition to their effects on the electrical excitability of neurons, neuromodulators also exert biochemical changes that are thought to be crucial for adaptive behaviors. In the striatum, within one projection neuron, different neuromodulators have been proposed to competitively and bidirectionally regulate synaptic transmission and plasticity via modulation of protein kinase A (PKA) activity. Between direct and indirect striatal pathways, the same neuromodulator can lead to either opposite (e.g. dopamine) or the same (e.g. opioid) direction of change of PKA activity in the projection neurons, thereby coordinating the two pathways that are implicated to produce opposite behavioral outcomes. Since neuromodulator inputs are constantly changing during animal behavior, how PKA responds to these dynamic and diverse inputs are crucial to understanding neuromodulator action. Nevertheless, the nature of this dynamic response is poorly understood. One of the major challenges to understanding this question is the lack of a dynamic readout of PKA activity in response to ongoing neuromodulatory inputs. Given the role of PKA as a major integrator of neuromodulator action and regulator of circuit plasticity, I have recently developed a PKA activity biosensor that can be targeted to genetically defined neurons for 2-photon Fluorescence Lifetime Imaging Microscopy (2pFLIM) in live brain slices. To understand neuromodulator action in the striatum, I propose to image PKA activity in genetically identified neurons with high spatiotemporal precision. First, given the importance of dopamine in normal striatal function (e.g. modulating movement and reward-based learning) and pathological conditions (e.g. Parkinson's Disease and addiction), I will characterize the dynamics of PKA activity in direct and indirect pathway striatal neurons in response to dopamine release. Secondly, I will determine how dopamine interacts with other modulators such as acetylcholine and opioids to alter PKA activity. In summary, this study will reveal how intracellular biochemical changes dynamically respond to ongoing neuromodulatory inputs, and how neuromodulators coordinate circuit dynamics. Understanding the integration and dynamics of neuromodulator action in normal conditions will shed light onto how abnormal neuromodulator signaling alters striatal function in pathological conditions such as neurodegenerative diseases and addiction.